SMB process for producing highly pure EPA from fish oil

ABSTRACT

The present invention provides a chromatographic separation process for recovering a polyunsaturated fatty acid (PUFA) product from a feed mixture which is a fish oil or which is derived from fish oil, which process comprises the steps of:
     (i) purifying the feed mixture in a chromatographic separation step, to obtain a first intermediate product; and   (ii) purifying the first intermediate product obtained in (i) in a simulated or actual moving bed chromatographic separation step, to obtain a second intermediate product; and   (iii) purifying the second intermediate product obtained in (ii) in a simulated or actual moving bed chromatographic separation step, to obtain the PUFA product; wherein an aqueous organic solvent is used as eluent in each separation step;   saturated and/or monounsaturated fatty acids present in the feed mixture are removed in the first separation step;   the PUFA product is separated from different components of the feed mixture in steps (ii) and (iii); and   the PUFA product obtained in the third separation step contains EPA or an EPA derivative in an amount greater than 90 wt %.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of co-pending U.S. application Ser.No. 13/880,146, filed Jul. 22, 2013, which is the National Phase entryof International Application No. PCT/GB2012/051593, filed Jul. 6, 2012,which claims priority to Great Britain Application No. 1111595.3, filedJul. 6, 2011, the disclosures of which are incorporated herein in theirentireties.

BACKGROUND OF THE INVENTION

The present invention relates to an improved chromatographic separationprocess for purifying the polyunsaturated fatty acid EPA or a derivativethereof.

EPA and its derivatives are precursors for biologically importantmolecules, which play an important role in the regulation of biologicalfunctions such as platelet aggregation, inflammation and immunologicalresponses. Thus, EPA and its derivatives may be therapeutically usefulin treating a wide range of pathological conditions including CNSconditions; neuropathies, including diabetic neuropathy; cardiovasculardiseases; general immune system and inflammatory conditions, includinginflammatory skin diseases.

EPA is found in natural raw materials, and in particular fish oils. TheEPA in fish oils is, however, present in such oils in admixture withsaturated fatty acids and numerous other impurities.

Purification of EPA from fish oils is particularly challenging. Thus,fish oils are extremely complex mixtures containing a large number ofdifferent components with very similar retention times in chromatographyapparatuses. They represent a much more challenging feedstock from whichto purify EPA than, for example, an algal oil feedstock. However, a veryhigh degree of purity of EPA is required, particularly forpharmaceutical and nutraceutical applications. Historically, therefore,distillation has been used to purify EPA for therapeutic applications.

Unfortunately, EPA is extremely fragile. Thus, when heated in thepresence of oxygen, it is prone to isomerization, peroxidation andoligomerization. The fractionation and purification of EPA to preparepure fatty acids is therefore difficult. Distillation, even undervacuum, can lead to non-acceptable product degradation.

Simulated and actual moving bed chromatography are known techniques,familiar to those of skill in the art. The principle of operationinvolves countercurrent movement of a liquid eluent phase and a solidadsorbent phase. This operation allows minimal usage of solvent makingthe process economically viable. Such separation technology has foundseveral applications in diverse areas, including hydrocarbons,industrial chemicals, oils, sugars and APIs.

As is well known, in a conventional stationary bed chromatographicsystem, a mixture whose components are to be separated percolatesthrough a container. The container is generally cylindrical, and istypically referred to as the column. The column contains a packing of aporous material (generally called the stationary phase) exhibiting ahigh permeability to fluids. The percolation velocity of each componentof the mixture depends on the physical properties of that component sothat the components exit from the column successively and selectively.Thus, some of the components tend to fix strongly to the stationaryphase and thus will percolate slowly, whereas others tend to fix weaklyand exit from the column more quickly. Many different stationary bedchromatographic systems have been proposed and are used for bothanalytical and industrial production purposes.

In contrast, a simulated moving bed chromatography apparatus consists ofa number of individual columns containing adsorbent which are connectedtogether in series. Eluent is passed through the columns in a firstdirection. The injection points of the feedstock and the eluent, and theseparated component collection points in the system, are periodicallyshifted by means of a series of valves. The overall effect is tosimulate the operation of a single column containing a moving bed of thesolid adsorbent, the solid adsorbent moving in a countercurrentdirection to the flow of eluent. Thus, a simulated moving bed systemconsists of columns which, as in a conventional stationary bed system,contain stationary beds of solid adsorbent through which eluent ispassed, but in a simulated moving bed system the operation is such as tosimulate a continuous countercurrent moving bed.

Processes and equipment for simulated moving bed chromatography aredescribed in several patents, including U.S. Pat. No. 2,985,589, U.S.Pat. No. 3,696,107, U.S. Pat. No. 3,706,812, U.S. Pat. No. 3,761,533,FR-A-2103302, FR-A-2651148 and FR-A-2651149, the entirety of which areincorporated herein by reference. The topic is also dealt with at lengthin “Preparative and Production Scale Chromatography”, edited by Ganetsosand Barker, Marcel Dekker Inc, New York, 1993, the entirety of which isincorporated herein by reference.

An actual moving bed system is similar in operation to a simulatedmoving bed system. However, rather than shifting the injection points ofthe feed mixture and the eluent, and the separated component collectionpoints by means of a system of valves, instead a series of adsorptionunits (i.e. columns) are physically moved relative to the feed anddrawoff points. Again, operation is such as to simulate a continuouscountercurrent moving bed.

Processes and equipment for actual moving bed chromatography aredescribed in several patents, including U.S. Pat. No. 6,979,402, U.S.Pat. No. 5,069,883 and U.S. Pat. No. 4,764,276, the entirety of whichare incorporated herein by reference.

A typical simulated moving bed chromatography apparatus is illustratedwith reference to FIG. 1. The concept of a simulated or actual movingbed chromatographic separation process is explained by considering avertical chromatographic column containing stationary phase S dividedinto sections, more precisely into four superimposed sub-zones I, II,III and IV going from the bottom to the top of the column. The eluent isintroduced at the bottom at IE by means of a pump P. The mixture of thecomponents A and B which are to be separated is introduced at IA+Bbetween sub-zone II and sub-zone III. An extract containing mainly B iscollected at SB between sub-zone I and sub-zone II, and a raffinatecontaining mainly A is collected at SA between sub-zone III and sub-zoneIV.

In the case of a simulated moving bed system, a simulated downwardmovement of the stationary phase S is caused by movement of theintroduction and collection points relative to the solid phase. In thecase of an actual moving bed system, simulated downward movement of thestationary phase S is caused by movement of the various chromatographiccolumns relative to the introduction and collection points. In FIG. 1,eluent flows upward and mixture A+B is injected between sub-zone II andsub-zone III. The components will move according to theirchromatographic interactions with the stationary phase, for exampleadsorption on a porous medium. The component B that exhibits strongeraffinity to the stationary phase (the slower running component) will bemore slowly entrained by the eluent and will follow it with delay. Thecomponent A that exhibits the weaker affinity to the stationary phase(the faster running component) will be easily entrained by the eluent.If the right set of parameters, especially the flow rate in eachsub-zone, are correctly estimated and controlled, the component Aexhibiting the weaker affinity to the stationary phase will be collectedbetween sub-zone III and sub-zone IV as a raffinate and the component Bexhibiting the stronger affinity to the stationary phase will becollected between sub-zone I and sub-zone II as an extract.

To achieve high purity EPA or EPA ethyl ester in purities of greaterthan 90%, for example greater than 95 or 97%, it is possible to utilisea simulated moving bed separation process which performs twosimultaneous separation steps. Such a process is described ininternational patent application no. PCT/GB10/002339, the entirety ofwhich is incorporated herein by reference.

In general, all chromatographic separation techniques for separatingPUFAs, including SMB processes, utilise large volumes of organicsolvents as eluents. After the chromatographic separation process iscompleted the PUFAs must be recovered from solution in the eluent.Typically a large expenditure of time and energy is involved inrecovering PUFAs from solution in the eluent. Furthermore, organicsolvents used as eluents in chromatographic separation processes arefrequently harmful to the environment or to the operatives handlingthem. Therefore, a chromatographic separation process which reduces theamount of organic solvent that needs to be used is required.

It has now been advantageously found that EPA or an EPA derivative canbe produced in a similarly high purity as described in PCT/GB10/002339by a three-step separation process which uses a much lower volume ofsolvent that the two-step process. The improved process of the presentinvention utilises almost 50% less solvent than the two-step processdescribed in PCT/GB10/002339. This is clearly advantageous in terms ofcost, ease of recovery of product, and environmental impact.

SUMMARY OF THE INVENTION

It has been surprisingly found that EPA or an EPA derivative can beeffectively purified from commercially available feedstocks such as fishoils by simulated or actual moving bed apparatus using a relatively lowvolume of an aqueous organic solvent eluent. The present inventiontherefore provides a chromatographic separation process for recovering apolyunsaturated fatty acid (PUFA) product from a feed mixture which is afish oil or which is derived from fish oil, which process comprises thesteps of:

-   (i) purifying the feed mixture in a chromatographic separation step,    to obtain a first intermediate product; and-   (ii) purifying the first intermediate product obtained in (i) in a    simulated or actual moving bed chromatographic separation step, to    obtain a second intermediate product; and-   (iii) purifying the second intermediate product obtained in (ii) in    a simulated or actual moving bed chromatographic separation step, to    obtain the PUFA product; wherein an aqueous organic solvent is used    as eluent in each separation step;-   saturated and/or monounsaturated fatty acids present in the feed    mixture are removed in the first separation step;-   the PUFA product is separated from different components of the feed    mixture in steps (ii) and (iii); and-   the PUFA product obtained in the third separation step contains EPA    or an EPA derivative in an amount greater than 90 wt %.

Also provided is a PUFA product obtainable by the process of the presentinvention.

DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the basic principles of a simulated or actual movingbed process for separating a binary mixture.

FIG. 2 illustrates three ways in which the chromatographic separationprocess of the invention may be carried out.

FIG. 3 illustrates a preferred embodiment of the invention which issuitable for producing high purity EPA.

FIG. 4 illustrates in more detail the embodiment of FIG. 2.

FIG. 5 illustrates a more preferred embodiment of the embodiment shownin FIG. 2.

FIG. 6 illustrates a two-stage separation process for producing EPA (notin accordance with the present invention).

FIG. 7 shows a GC trace of a suitable feed stock for use in accordancewith the process of the present invention.

FIG. 8 shows a GC trace of a first intermediate product produced inaccordance with the process of the present invention.

FIG. 9 shows a GC trace of a second intermediate product produced inaccordance with the process of the present invention.

FIG. 10 shows a GC trace of a PUFA product produced in accordance withthe process of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “PUFA product” refers to a product comprisingone or more polyunsaturated fatty acids (PUFAs), and/or derivativesthereof, typically of nutritional or pharmaceutical significance. ThePUFA product obtained in the process of the present invention containsEPA or an EPA derivative in an amount greater than 90 wt %, i.e. EPA oran EPA derivative is present at 90 wt % purity relative to all of thecomponents in the final PUFA product not including the aqueous organicsolvent eluent. Thus EPA or an EPA derivative is present in the PUFAproduct in an amount of at least 90 wt % on the basis of all of thecomponents of the PUFA product which originated in the feed mixture.

An EPA derivative is EPA in the form of a mono-, di- or tri-glyceride,ester, phospholipid, amide, lactone, or salt. Triglycerides and estersare preferred. Esters are more preferred. Esters are typically alkylesters, preferably C₁-C₆ alkyl esters, more preferably C₁-C₄ alkylesters. Examples of esters include methyl and ethyl esters. Ethyl estersare most preferred.

Typically, the PUFA product contains EPA or an EPA derivative in anamount greater than 95 wt %, preferably greater than 97 wt %.

In one embodiment, the PUFA product contains EPA in an amount greaterthan 90 wt %, preferably greater than 95 wt %, more preferably greaterthan 97 wt %. As explained above, EPA is present at the specified wt %relative to the total amount of all of the components of the PUFAproduct which originated in the feed mixture.

In another embodiment, the PUFA product contains EPA ethyl ester in anamount greater than 90 wt %, preferably greater than 95 wt %, morepreferably greater than 97 wt %. As explained above, EPA is present atthe specified wt % relative to the total amount of all of the componentsof the PUFA product which originated in the feed mixture.

Suitable feed mixtures for fractionating by the process of the presentinvention are fish oils, or feed stocks derived from fish oils. Suitablefish oils for use in the process of the present invention are well knownto the skilled person. Typical fish oils contain EPA, DHA, SDA, andtypically a range of other PUFAs both more and less polar than EPA,saturated fatty acids and monounsaturated fatty acids.

The feed mixture may undergo chemical treatment before fractionation bythe process of the invention. For example, it may undergo glyceridetransesterification or glyceride hydrolysis followed in certain cases byselective processes such as crystallisation, molecular distillation,urea fractionation, extraction with silver nitrate or other metal saltsolutions, iodolactonisation or supercritical fluid fractionation.Alternatively, a feed mixture may be used directly with no initialtreatment step.

The feed mixtures typically contain the PUFA product and at least onemore polar component and at least one less polar component. The lesspolar components have a stronger adherence to the adsorbent used in theprocess of the present invention than does the PUFA product. Duringoperation, such less polar components typically move with the solidadsorbent phase in preference to the liquid eluent phase. The more polarcomponents have a weaker adherence to the adsorbent used in the processof the present invention than does the PUFA product. During operation,such more polar components typically move with the liquid eluent phasein preference to the solid adsorbent phase. In general, more polarcomponents will be separated into a raffinate stream, and less polarcomponents will be separated into an extract stream.

Examples of the more and less polar components include (1) othercompounds occurring in natural oils (e.g. marine oils), (2) byproductsformed during storage, refining and previous concentration steps and (3)contaminants from solvents or reagents which are utilized duringprevious concentration or purification steps.

Examples of (1) include other unwanted PUFAs; saturated fatty acids;sterols, for example cholesterol; vitamins; and environmentalpollutants, such as polychlorobiphenyl (PCB), polyaromatic hydrocarbon(PAH) pesticides, chlorinated pesticides, dioxines and heavy metals.PCB, PAH, dioxines and chlorinated pesticides are all highly non-polarcomponents.

Examples of (2) include isomers and oxidation or decomposition productsfrom the PUFA product, for instance, auto-oxidation polymeric productsof fatty acids or their derivatives.

Examples of (3) include urea which may be added to remove saturated ormonounsaturated fatty acids from the feed mixture.

Preferably, the feed mixture is a PUFA-containing marine oil (e.g. afish oil), more preferably a marine oil (e.g. a fish oil) comprising EPAand/or DHA.

A typical feed mixture for preparing concentrated EPA (EE) by theprocess of the present invention comprises 50-75% EPA (EE), 0 to 10% DHA(EE), and other components including other essential ω-3 and ω-6 fattyacids.

A preferred feed mixture for preparing concentrated EPA (EE) by theprocess of the present invention comprises 55% EPA (EE), 5% DHA (EE),and other components including other essential ω-3 and ω-6 fatty acids.DHA (EE) is less polar than EPA (EE).

The process of the present invention involves multiple chromatographyseparation steps.

The first separation step is effective to remove saturated and/ormonounsaturated fatty acids present in the feed mixture and may becarried out using a stationary bed or simulated or actual moving bedchromatography apparatus.

When the first separation step comprises purifying the feed mixture in asimulated or actual moving bed chromatography apparatus, there areseveral ways in which the three separation steps may be realised. Fourpreferred ways of carrying out the process are given as first, second,third and fourth embodiments below.

In a first embodiment, the first, second and third separation steps arecarried out simultaneously in a single simulated or actual moving bedchromatography apparatus having a plurality of linked chromatographycolumns containing, as eluent, an aqueous organic solvent, the first,second and third separation steps being carried out in first, second andthird zones respectively, wherein each zone has one or more injectionpoints for a feed mixture stream, one or more injection points for waterand/or organic solvent, a raffinate take-off stream from which liquidcan be collected from said zone, and an extract take-off stream fromwhich liquid can be collected from said zone.

Typically, each zone has only one injection point for a feed mixture. Inone embodiment, each zone has only one injection point for the aqueousorganic solvent eluent. In another embodiment, each zone has two or moreinjection points for water and/or organic solvent.

Typically each zone used has a single array of chromatography columnslinked in series containing, as eluent, an aqueous organic solvent.Typically, each of the chromatography columns in a zone are linked tothe two columns in the apparatus adjacent to that column. Thus, theoutput from a given column in a zone is connected to the input of theadjacent column, for example in the zone, which is downstream withrespect to the flow of eluent in the system. Typically, none of thechromatography columns in a zone are linked to non-adjacent columns inthe same zone.

The term “raffinate” is well known to the person skilled in the art. Inthe context of actual and simulated moving bed chromatography it refersto the stream of components that move more rapidly with the liquideluent phase compared with the solid adsorbent phase. Thus, a raffinatestream is typically enriched with more polar components, and depleted ofless polar components compared with a feed stream.

The term “extract” is well known to the person skilled in the art. Inthe context of actual and simulated moving bed chromatography it refersto the stream of components that move more rapidly with the solidadsorbent phase compared with the liquid eluent phase. Thus, an extractstream is typically enriched with less polar components, and depleted ofmore polar components compared with a feed stream.

As used herein the term “nonadjacent” refers to columns, in for examplethe same apparatus, separated by one or more columns, preferably 3 ormore columns, more preferably 5 or more columns, most preferably about 5columns.

In a second embodiment, the first and second separation steps arecarried out simultaneously in a single simulated or actual moving bedchromatography apparatus having a plurality of linked chromatographycolumns containing, as eluent, an aqueous organic solvent, the first andsecond separation steps being carried out in first and second zonesrespectively, wherein each zone is as defined herein, and wherein thethird separation step is carried out in a separate simulated or actualmoving bed chromatography apparatus.

In the second embodiment, the third separation step is typically carriedout in a simulated or actual moving bed chromatography apparatuscomprising a plurality of linked chromatography columns containing, aseluent, an aqueous organic solvent, and having one or more injectionpoints for a feed mixture stream, one or more injection points for waterand/or organic solvent, a raffinate take-off stream from which liquidcan be collected from said plurality of linked chromatography columns,and an extract take-off stream from which liquid can be collected fromsaid plurality of linked chromatography columns. This chromatographyapparatus typically has only one injection point for a feed mixture. Inone embodiment, this chromatography apparatus has only one injectionpoint for the aqueous organic solvent eluent. In another embodiment,this chromatography apparatus has two or more injection points for waterand/or organic solvent.

The chromatography apparatus used in the third separation step in thesecond embodiment typically has a single array of chromatography columnslinked in series containing, as eluent, an aqueous organic solvent.Typically, each of the chromatography columns are linked to the twocolumns in the apparatus adjacent to that column. Thus, the output froma given column is connected to the input of the adjacent column, whichis downstream with respect to the flow of eluent in the system.Typically, none of the chromatography columns are linked to non-adjacentcolumns in the chromatography apparatus.

The chromatography apparatus used in the third separation step in thesecond embodiment is a separate apparatus from the apparatus used in thefirst and second separation steps. Thus, two separate apparatuses areused. Eluent circulates separately in the separate chromatographicapparatuses. Thus, eluent is not shared between the separatechromatographic apparatuses other than what eluent may be present assolvent in the second intermediate product which is produced in thesecond step, and which is then introduced into the chromatographicapparatus used in the third separation step. Chromatographic columns arenot shared between the separate chromatographic apparatuses.

After the second intermediate product is obtained in the secondseparation step, the aqueous organic solvent eluent may be partly ortotally removed before the second intermediate product is purifiedfurther in the third separation step. Alternatively, the intermediateproduct may be purified further in the third step without the removal ofany solvent present.

The chromatography apparatus used in the third separation step in thesecond embodiment is similar to the chromatography apparatus illustratedin FIG. 1.

In a third embodiment, the second and third separation steps are carriedout simultaneously in a single simulated or actual moving bedchromatography apparatus having a plurality of linked chromatographycolumns containing, as eluent, an aqueous organic solvent, the secondand third separation steps being carried out in first and second zonesrespectively, wherein each zone is as defined herein, and wherein thefirst separation step is carried out in a separate simulated or actualmoving bed chromatography apparatus.

In the third embodiment, the first separation step is typically carriedout in a simulated or actual moving bed chromatography apparatuscomprising a plurality of linked chromatography columns containing, aseluent, an aqueous organic solvent, and having one or more injectionpoints for a feed mixture stream, one or more injection points for waterand/or organic solvent, a raffinate take-off stream from which liquidcan be collected from said plurality of linked chromatography columns,and an extract take-off stream from which liquid can be collected fromsaid plurality of linked chromatography columns. This chromatographyapparatus typically has only one injection point for a feed mixture. Inone embodiment, this chromatography apparatus has only one injectionpoint for the aqueous organic solvent eluent. In another embodiment,this chromatography apparatus has two or more injection points for waterand/or organic solvent.

The chromatography apparatus used in the first separation step in thethird embodiment typically has a single array of chromatography columnslinked in series containing, as eluent, an aqueous organic solvent.Typically, each of the chromatography columns are linked to the twocolumns in the apparatus adjacent to that column. Thus, the output froma given column is connected to the input of the adjacent column, whichis downstream with respect to the flow of eluent in the system.Typically, none of the chromatography columns are linked to non-adjacentcolumns in the chromatography apparatus.

The chromatography apparatus used in the first separation step in thethird embodiment is a separate apparatus from the apparatus used in thesecond and third separation steps. Thus, two separate apparatuses areused. Eluent is not shared between the separate chromatographicapparatuses other than what eluent may be present as solvent in thefirst intermediate product which is produced in the first step, andwhich is introduced into the chromatographic apparatus used in thesecond separation step. Chromatographic columns are not shared betweenthe separate chromatographic apparatuses.

After the first intermediate product is obtained in the first separationstep, the aqueous organic solvent eluent may be partly or totallyremoved before the intermediate product is purified further in the nextseparation step. Alternatively, the first intermediate product may bepurified further in the second separation step without the removal ofany solvent present.

The chromatography apparatus used in the first separation step in thethird embodiment is similar to the chromatography apparatus illustratedin FIG. 1.

It will be appreciated that in the first, second and third embodimentsabove two or more separation steps may take place simultaneously in asingle simulated or actual moving bed chromatography apparatus havingtwo or three zones, wherein a zone is as defined above. A typicalchromatography apparatus having two or more zones, for example two orthree zones, is as described in, for example, PCT/GB10/002339, which isincorporated herein by reference.

In a fourth embodiment, either (a) the first, second and thirdseparation steps are carried out sequentially on the same chromatographyapparatus, first and second intermediate products being recoveredbetween the first and second, and second and third separation stepsrespectively, and the process conditions in the chromatography apparatusbeing adjusted between the first and second, and second and thirdseparation steps such that saturated and/or monounsaturated fatty acidspresent in the feed mixture are removed in the first separation step,and the PUFA product is separated from different components of the feedmixture in steps (ii) and (iii); or (b) the second separation step iscarried out using a different chromatographic apparatus to that used inthe first separation step, and/or the third separation step is carriedout using a different chromatographic apparatus to that used in thesecond separation step.

In the fourth embodiment, each of the chromatography apparatuses used tocarry out the first, second and third separation steps is typically asdefined above for the third separation step in embodiment (2).

In option (b) of the fourth embodiment, all three steps are carried outon separate chromatographic apparatuses. Two or three of the first,second and third separation steps are carried out on two or threedifferent separate chromatographic apparatuses. These may be operatedsequentially or simultaneously.

In particular, in option (b) of the fourth embodiment two separatechromatography apparatuses may be operated sequentially to carry out thefirst and second separation steps. In this case, the first intermediateproduct is recovered between the first and second separation steps andthe process conditions in the first and second chromatographyapparatuses are adjusted such that saturated and/or monounsaturatedfatty acids present in the feed mixture are removed in the firstseparation step and the PUFA product is separated from differentcomponents of the feed mixture in steps (ii) and (iii).

In particular, in option (b) of the fourth embodiment two separatechromatography apparatuses may be operated sequentially to carry out thesecond and third separation steps. In this case, the second intermediateproduct is recovered between the second and third separation steps andthe process conditions in the second and third chromatographyapparatuses are adjusted such that saturated and/or monounsaturatedfatty acids present in the feed mixture are removed in the firstseparation step and the PUFA product is separated from differentcomponents of the feed mixture in steps (ii) and (iii).

In particular, in option (b) of the fourth embodiment three separatechromatography apparatuses may be operated sequentially to carry out thefirst, second and third separation steps. In this case, the firstintermediate product is recovered between the first and secondseparation steps, the second intermediate product is recovered betweenthe second and third separation steps and the process conditions in thefirst, second and third chromatography apparatuses are adjusted suchthat saturated and/or monounsaturated fatty acids present in the feedmixture are removed in the first separation step and the PUFA product isseparated from different components of the feed mixture in steps (ii)and (iii).

In particular, in option (b) of the fourth embodiment, two separatechromatography apparatuses may be operated simultaneously to carry outthe first and second separation steps. The first and second separationsteps are carried out on separate chromatography apparatuses, the firstintermediate product obtained in the first step being introduced intothe chromatography apparatus used in the second separation step, and theprocess conditions in the chromatography apparatuses being adjusted suchthat saturated and/or monounsaturated fatty acids present in the feedmixture are removed in the first separation step and the PUFA product isseparated from different components of the feed mixture in steps (ii)and (iii).

In particular, in option (b) of the fourth embodiment, two separatechromatography apparatuses may be operated simultaneously to carry outthe second and third separation steps. The second and third separationsteps are carried out on separate chromatography apparatuses, the secondintermediate product obtained in the second step being introduced intothe chromatography apparatus used in the third separation step, and theprocess conditions in the chromatography apparatuses being adjusted suchthat saturated and/or monounsaturated fatty acids present in the feedmixture are removed in the first separation step and the PUFA product isseparated from different components of the feed mixture in steps (ii)and (iii).

In particular, in option (b) of the fourth embodiment, three separatechromatography apparatuses may be operated simultaneously to carry outthe first, second and third separation steps. The first, second andthird separation steps are carried out on separate chromatographyapparatuses, the first intermediate product obtained in the first stepbeing introduced into the chromatography apparatus used in the secondseparation step, the second intermediate product obtained in the secondstep being introduced into the chromatography apparatus used in thethird separation step, and the process conditions in the chromatographyapparatuses being adjusted such that saturated and/or monounsaturatedfatty acids present in the feed mixture are removed in the firstseparation step and the PUFA product is separated from differentcomponents of the feed mixture in steps (ii) and (iii).

In particular, in option (b) of the fourth embodiment, two or threeseparate chromatographic apparatuses are operated. Eluent circulatesseparately in the separate chromatographic apparatuses. Thus, eluent isnot shared between the separate chromatographic apparatuses other thanwhat eluent may be present as solvent in the intermediate product whichis purified in the first and/or second step, and which is introducedinto the chromatographic apparatus used in the next separation step.Chromatographic columns are not shared between the separatechromatographic apparatuses used in the first and second and/or secondand third separation steps.

After the intermediate product is obtained in the first and/or secondseparation step, the aqueous organic solvent eluent may be partly ortotally removed before the intermediate product is purified further inthe next separation step. Alternatively, the intermediate product may bepurified further without the removal of any solvent present. Theseconsiderations also apply for the second intermediate product obtainedin the second separation step in embodiment (2) above, and for the firstintermediate product obtained in the first separation step in embodiment(3) above.

In general, any known stationary bed or simulated or actual moving bedchromatography apparatus may be utilised for the purposes of the methodof the present invention, as long as the apparatus is used in accordancewith the process of the present invention. Those apparatuses describedin PCT/GB10/002339, U.S. Pat. No. 2,985,589, U.S. Pat. No. 3,696,107,U.S. Pat. No. 3,706,812, U.S. Pat. No. 3,761,533, FR-A-2103302,FR-A-2651148, FR-A-2651149, U.S. Pat. No. 6,979,402, U.S. Pat. No.5,069,883 and U.S. Pat. No. 4,764,276 may all be used if configured inaccordance with the process of the present invention.

The second, third and fourth embodiments above are preferred. The thirdand fourth embodiments are more preferred. For certain applications, thethird embodiment will be most suitable. In other applications, thefourth embodiment will be most suitable.

The first to fourth embodiments are illustrated in more detail withreference to FIG. 2. In all four embodiments in FIG. 2 the flow ofeluent is from right to left, and the effective flow of adsorbent isfrom left to right. It can be seen in all cases that the firstintermediate product obtained from the first separation step is used asthe feed mixture for the second separation step, and the secondintermediate product is used as the feed mixture for the thirdseparation step.

Referring now to FIG. 2A, this illustrates the first embodiment above,i.e. where the first, second and third separation steps are carried outin a single simulated or actual moving bed chromatography apparatus infirst, second and third zones respectively. The first separation steptakes place in the first zone. Then the first intermediate product fromthe first separation step carried out in the first zone is passed intothe second zone as the feed mixture. The second separation step is thencarried out in the second zone. The second intermediate product is thenpassed from the second separation step carried out in the second zoneinto the third zone as the feed mixture. The third separation step isthen carried out in the third zone.

Referring now to FIG. 2B, this illustrates the second embodiment above,i.e. where the first and second separation steps are carried outsimultaneously in a single simulated or actual moving bed chromatographyapparatus in first and second zones respectively, and the thirdseparation step is carried out in a separate simulated or actual movingbed chromatography apparatus. The first separation step takes place inthe first zone. Then the first intermediate product from the firstseparation step carried out in the first zone is passed into the secondzone as the feed mixture. The second separation step is carried out inthe second zone. The second intermediate product is collected from thesecond zone. This is then introduced into a chromatography apparatus asthe feed mixture for the third separation step.

Referring now to FIG. 2C, this illustrates the third embodiment above,i.e. where the second and third separation steps are carried outsimultaneously in a single simulated or actual moving bed chromatographyapparatus in first and second zones respectively, and the firstseparation step is carried out in a separate simulated or actual movingbed chromatography apparatus. The first separation step takes place in achromatography apparatus. The first intermediate product is collectedfrom the first apparatus. This is then introduced into a separatechromatography apparatus as the feed mixture for the second separationstep. The second separation step is carried out in the first zone of thechromatographic apparatus in which the second and third separation stepstake place. The second intermediate product from the second separationstep carried out in the first zone is passed into the second zone as thefeed mixture for the third separation step. The third separation steptakes place in the second zone.

Referring now to FIG. 2D, this illustrates the fourth embodiment above,i.e. where (a) the first, second and third separation steps are carriedout sequentially on the same chromatography apparatus, first and secondintermediate products being recovered between the first and second, andsecond and third separation steps respectively, and the processconditions in the chromatography apparatus being adjusted between thefirst and second, and second and third separation steps such thatsaturated and/or monounsaturated fatty acids present in the feed mixtureare removed in the first separation step, and the PUFA product isseparated from different components of the feed mixture in steps (ii)and (iii); or (b) two or three of the first, second and third separationsteps are carried out on two or three different separate apparatuses;wherein the second separation step is carried out using a differentchromatographic apparatus to that used in the first separation step,and/or the third separation step is carried out using a differentchromatographic apparatus to that used in the second separation step.

When the first separation step comprises purifying the feed mixture in astationary bed chromatography apparatus, there are several ways in whichthe three separation steps may be realised. Thus typically, (a) thesecond and third separation steps are carried out simultaneously in asingle simulated or actual moving bed chromatography apparatus having aplurality of linked chromatography columns containing, as eluent, anaqueous organic solvent, the second and third separation steps beingcarried out in first and second zones respectively, wherein each zone isas defined herein; or

-   (b) the second and third separation steps are carried out    sequentially on the same chromatography apparatus, the second    intermediate product being recovered between the second and third    separation steps and the process conditions in the chromatography    apparatus being adjusted between the second and third separation    steps such that the PUFA product is separated from different    components of the feed mixture in steps (ii) and (iii); or-   (c) the second and third separation steps are carried out on    separate chromatography apparatuses respectively, the intermediate    product obtained from the second separation step being introduced    into the chromatography apparatus used in the third separation step.

Embodiment (a) above is carried out in a similar manner to the secondand third separation steps in embodiment (3) above.

The chromatography apparatuses used in embodiments (b) and (c) above istypically as defined above for the third separation step in embodiment(2). Embodiments (b) and (c) are typically carried out in a similarmanner to embodiment (4) above.

It will be appreciated that in certain embodiments, two or threeseparation steps may be carried out simultaneously in a singlechromatography apparatus having two or three zones respectively. Insimulated or actual moving bed chromatography apparatuses in which twoseparation steps are carried out simultaneously in two zones, araffinate or extract stream is typically collected from a column in thefirst zone and introduced to a nonadjacent column in the second zone. Insimulated or actual moving bed chromatography apparatuses in which threeseparation steps are carried out simultaneously in three zones, araffinate or extract stream is typically collected from a column in thefirst zone and introduced to a nonadjacent column in the second zone,and a raffinate or extract stream is typically collected from a columnin the second zone and introduced to a nonadjacent column in the thirdzone. This enables the first and/or second intermediate productscollected in the first and/or second separation steps to be used as thefeed mixture for the next separation step.

Typically, the second intermediate product is collected as the raffinatestream in the second separation step, and the PUFA product is collectedas the extract stream in the third separation step; or the secondintermediate product is collected as the extract stream in the secondseparation step, and the PUFA product is collected as the raffinatestream in the third separation step.

Preferably, the second intermediate product is collected as theraffinate stream in the second separation step, and the PUFA product iscollected as the extract stream in the third separation step.

Typically, in embodiments where the second and third separation stepsare carried out simultaneously in a single simulated or actual movingbed chromatography apparatus in first and second zones respectively, (a)the second intermediate product is collected as a raffinate streamcontaining the PUFA product together with more polar components from acolumn in the first zone and introduced to a nonadjacent column in thesecond zone, where the PUFA product is then collected as the extractstream in the third separation step carried out in the second zone; or(b) the second intermediate product is collected as an extract streamcontaining the PUFA product together with less polar components from acolumn in the first zone and introduced to a nonadjacent column in thesecond zone, where the PUFA product is then collected as the raffinatestream in the third separation step carried out in the second zone.

Preferably, in embodiments where the second and third separation stepsare carried out simultaneously in a single simulated or actual movingbed chromatography apparatus in first and second zones respectively, thesecond intermediate product is collected as a raffinate streamcontaining the PUFA product together with more polar components from acolumn in the first zone and introduced to a nonadjacent column in thesecond zone, where the PUFA product is then collected as the extractstream in the third separation step which is carried out in the secondzone.

When the first separation step comprises purifying the feed mixture in asimulated or actual moving bed chromatography apparatus, the firstintermediate product is typically collected as the raffinate stream inthe first separation step.

When the first separation step comprises purifying the feed mixture in asimulated or actual moving bed chromatography apparatus, the firstintermediate product is typically collected as the raffinate stream inthe first separation step and (a) the second intermediate product iscollected as the raffinate stream in the second separation step, and thePUFA product is collected as the extract stream in the third separationstep; or (b) the second intermediate product is collected as the extractstream in the second separation step, and the PUFA product is collectedas the raffinate stream in the third separation step.

Typically, the first intermediate product obtained in the firstseparation step is enriched in the PUFA product compared to the feedmixture; and/or the second intermediate product obtained in the secondseparation step is enriched in the PUFA product compared to the firstintermediate product.

Preferably the first intermediate product obtained in the firstseparation step is enriched in the PUFA product compared to the feedmixture and the second intermediate product obtained in the secondseparation step is enriched in the PUFA product compared to the firstintermediate product.

Typically, the first intermediate product obtained in the firstseparation step is depleted in saturated and/or monounsaturated fattyacids compared to the feed mixture.

Typically, in the first step the PUFA product is separated fromcomponents of the feed mixture which are less polar than the PUFAproduct, in the second step the PUFA product is separated fromcomponents of the feed mixture which are less polar than the PUFAproduct but more polar than the components separated in the firstseparation step, and in the third separation step the PUFA product isseparated from components which are more polar than the PUFA product.

Alternatively, in the first step the PUFA product is separated fromcomponents of the feed mixture which are less polar than the PUFAproduct, in the second step the PUFA product is separated fromcomponents of the feed mixture which are more polar than the PUFAproduct, and in the third separation step the PUFA product is separatedfrom components which are less polar than the PUFA product but morepolar than the components separated in the first separation step.

The components of the feed mixture separated in the first step which areless polar than the PUFA product are typically unsaturated and/ormonounsaturated fatty acids.

The components of the feed mixture which are less polar than the PUFAproduct but more polar than the components separated in the firstseparation step typically include DHA or a DHA derivative and/or otherPUFAs or PUFA derivatives which are less polar than the PUFA product butmore polar than the components separated in the first separation step.

The components of the feed mixture which are more polar than the PUFAproduct include SDA or an SDA derivative and/or other PUFAs which aremore polar than the PUFA product.

PUFAs other than EPA are well known and include ω-3 and ω-6 PUFAs.Examples of ω-3 PUFAs include alpha-linolenic acid (ALA), stearidonicacid (SDA), eicosatrienoic acid (ETE), eicosatetraenoic acid (ETA),docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA). Examples ofω-6 PUFAs include linoleic acid (LA), gamma-linolenic acid (GLA),eicosadienoic acid, dihomo-gamma-linolenic acid (DGLA), arachidonic acid(ARA), docosadienoic acid, adrenic acid and docosapentaenoic (ω-6) acid.

The number of columns used in each separation step is not particularlylimited. A skilled person would easily be able to determine anappropriate number of columns to use. The number of columns is typically4 or more, preferably 6 or more, more preferably 8 or more, for example4, 5, 6, 7, 8, 9, or 10 columns. In preferred embodiment, 5 or 6columns, more preferably 6 columns are used. In another preferredembodiment, 7 or 8 columns, more preferably 8 columns are used.Typically, there are no more than 25 columns, preferably no more than20, more preferably no more than 15.

In embodiments where two separation steps take place simultaneously in asingle chromatography apparatus in first and second zones respectively,the number of columns in each zone is typically 4 or more, preferably 6or more, more preferably 8 or more, for example 4, 5, 6, 7, 8, 9, or 10columns.

In embodiments where three separation steps take place simultaneously ina single chromatography apparatus in first, second and third zonesrespectively, the number of columns in each zone is typically 4 or more,preferably 6 or more, more preferably 8 or more, for example 4, 5, 6, 7,8, 9, or 10 columns.

The first, second and third separation steps typically involve the samenumber of columns. For certain applications they may have differentnumbers of columns.

The dimensions of the columns used are not particularly limited, andwill depend on the volume of feed mixture to be purified. A skilledperson would easily be able to determine appropriately sized columns touse. The diameter of each column is typically between 10 and 1000 mm,preferably between 10 and 500 mm, more preferably between 25 and 250 mm,even more preferably between 50 and 100 mm, and most preferably between70 and 80 mm. The length of each column is typically between 10 and 300cm, preferably between 10 and 200 cm, more preferably between 25 and 150cm, even more preferably between 70 and 110 cm, and most preferablybetween 80 and 100 cm.

The first, second and third separation steps typically involve columnshaving identical dimensions but may, for certain applications, they havedifferent dimensions.

The flow rates to the column are limited by maximum pressures across theseries of columns and will depend on the column dimensions and particlesize of the solid phases. One skilled in the art will easily be able toestablish the required flow rate for each column dimension to ensureefficient desorption. Larger diameter columns will in general needhigher flows to maintain linear flow through the columns.

For the typical column sizes outlined above, typically the flow rate ofeluent into the chromatographic apparatus used in the first or secondseparation step is from 1 to 4.5 L/min, preferably from 1.5 to 2.5L/min. Typically, the flow rate of the extract from the chromatographicapparatus used in the first or second separation step is from 0.1 to 2.5L/min, preferably from 0.5 to 2.25 L/min. In embodiments where part ofthe extract from the first or second separation step is recycled backinto the apparatus used in the first or second separation step, the flowrate of recycle is typically from 0.7 to 1.4 L/min, preferably about 1L/min. Typically, the flow rate of the raffinate from thechromatographic apparatus used in the first or second separation step isfrom 0.2 to 2.5 L/min, preferably from 0.3 to 2.0 L/min. In embodimentswhere part of the raffinate from the first or second separation step isrecycled back into the apparatus used in the first or second separationstep, the flow rate of recycle is typically from 0.3 to 1.0 L/min,preferably about 0.5 L/min. Typically, the flow rate of introduction ofthe feed mixture into the chromatographic apparatus used in the first orsecond separation step is from 5 to 150 mL/min, preferably from 10 to100 mL/min, more preferably from 20 to 60 mL/min.

For the typical column sizes outlined above, typically the flow rate ofeluent into the chromatographic apparatus used in the third separationstep is from 1 to 4 L/min, preferably from 1.5 to 3.5 L/min. Typically,the flow rate of the extract from the chromatographic apparatus used inthe third separation step is from from 0.5 to 2 L/min, preferably from0.7 to 1.9 L/min. In embodiments where part of the extract from thethird separation step is recycled back into the apparatus used in thethird separation step, the flow rate of recycle is typically from 0.6 to1.4 L/min, preferably from 0.7 to 1.1 L/min, more preferably about 0.9L/min. Typically, the flow rate of the raffinate from thechromatographic apparatus used in the third separation step is from 0.5to 2.5 L/min, preferably from 0.7 to 1.8 L/min, more preferably about1.4 L/min. In embodiments where part of the raffinate from the thirdseparation step is recycled back into the apparatus used in the thirdseparation step, the flow rate of recycle is typically from 0.3 to 1.0L/min, preferably about 0.5 L/min.

As the skilled person will appreciate, references to rates at whichliquid is collected or removed via the various extract and raffinatestreams refer to volumes of liquid removed in an amount of time,typically L/minute. Similarly, references to rates at which liquid isrecycled back into an apparatus, typically to an adjacent column in theapparatus, refer to volumes of liquid recycled in an amount of time,typically L/minute.

The step time, i.e. the time between shifting the points of injection ofthe feed mixture and eluent, and the various take off points of thecollected fractions, is not particularly limited, and will depend on thenumber and dimensions of the columns used, and the flow rate through theapparatus. A skilled person would easily be able to determineappropriate step times to use in the process of the present invention.The step time is typically from 100 to 1000 seconds, preferably from 200to 800 seconds, more preferably from about 250 to about 750 seconds. Insome embodiments, a step time of from 100 to 400 seconds, preferably 200to 300 seconds, more preferably about 250 seconds, is appropriate. Inother embodiments, a step time of from 600 to 900 seconds, preferably700 to 800 seconds, more preferably about 750 seconds is appropriate.

In the process of the present invention, actual moving bedchromatography is preferred.

Conventional adsorbents known in the art for actual and simulated movingbed systems may be used in the process of the present invention. Eachchromatographic column may contain the same or a different adsorbent.Typically, each column contains the same adsorbent. Examples of suchcommonly used materials are polymeric beads, preferably polystyrenereticulated with DVB (divinylbenzene); and silica gel, preferablyreverse phase bonded silica gel with C8 or C18 alkanes, especially C18.C18 bonded reverse phase silica gel is preferred. The adsorbent used inthe process of the present invention is preferably non-polar.

The shape of the adsorbent stationary phase material may be, forexample, spherical or nonspherical beads, preferably substantiallyspherical beads. Such beads typically have a diameter of 5 to 500microns, preferably 10 to 500 microns, more preferably 15 to 500microns, more preferably 40 to 500 microns, more preferably 100 to 500microns, more preferably 250 to 500 microns, even more preferably 250 to400 microns, most preferably 250 to 350 microns. In some embodiments,beads with a diameter of 5 to 35 microns may be used, typically 10 to 30microns, preferably 15 to 25 microns. Some preferred particle sizes aresomewhat larger than particle sizes of beads used in the past insimulated and actual moving bed processes. Use of larger particlesenables a lower pressure of eluent to be used in the system. This, inturn, has advantages in terms of cost savings, efficiency and lifetimeof the apparatus. It has surprisingly been found that adsorbent beads oflarge particle size may be used in the process of the present invention(with their associated advantages) without any loss in resolution.

The adsorbent typically has a pore size of from 10 to 50 nm, preferably15 to 45 nm, more preferably 20 to 40 nm, most preferably 25 to 35 nm.

Typically, the process of the present invention is conducted at from 15to 55° C., preferably at from 20 to 40° C., more preferably at about 30°C. Thus, the process is typically carried out at room temperature, butmay be conducted at elevated temperatures.

As mentioned above, saturated and/or monounsaturated fatty acids presentin the feed mixture are removed in the first separation step and thePUFA product is separated from different components of the feed mixturein steps (ii) and (iii). This is typically effected by adjusting theprocess conditions in the chromatography apparatus, or zone in achromatography apparatus in which the first, second and third separationsteps are carried out.

Thus, the process conditions in the first, second and third separationsteps typically vary. The process conditions which vary may include, forexample, the size of the columns used, the number of columns used, thepacking used in the columns, the step time of the SMB apparatus, thetemperature of the apparatus, the eluent used in the separation steps,or the flow rates used in the apparatus, in particular the recycle rateof liquid collected via the extract or raffinate streams.

Preferably the process conditions which vary are the water:organicsolvent ratio of the eluent used in the separation steps, and/or therecycle rate of liquid collected via the extract or raffinate streams inthe separation steps. Both of these options are discussed in more detailbelow.

Typically, part of the extract stream from the apparatus used in thesecond separation step is recycled back into the apparatus used in thesecond separation step; and/or part of the raffinate stream from theapparatus used in the second separation step is recycled back into theapparatus used in the second separation step; and/or part of the extractstream from the apparatus used in the third separation step is recycledback into the apparatus used in the third separation step; and/or partof the raffinate stream from the apparatus used in the third separationstep is recycled back into the apparatus used in the third separationstep.

Preferably, part of the extract stream from the apparatus used in thesecond separation step is recycled back into the apparatus used in thesecond separation step; and part of the raffinate stream from theapparatus used in the second separation step is recycled back into theapparatus used in the second separation step; and part of the extractstream from the apparatus used in the third separation step is recycledback into the apparatus used in the third separation step; and part ofthe raffinate stream from the apparatus used in the third separationstep is recycled back into the apparatus used in the third separationstep.

When the first separation step comprises purifying the feed mixture in asimulated or actual moving bed chromatography apparatus, typically partof the extract stream from the apparatus used in the first separationstep is recycled back into the apparatus used in the first separationstep; and/or part of the raffinate stream from the apparatus used in thefirst separation step is recycled back into the apparatus used in thefirst separation step.

When the first separation step comprises purifying the feed mixture in asimulated or actual moving bed chromatography apparatus, preferably partof the extract stream from the apparatus used in the first separationstep is recycled back into the apparatus used in the first separationstep; and part of the raffinate stream from the apparatus used in thefirst separation step is recycled back into the apparatus used in thefirst separation step; and part of the extract stream from the apparatusused in the second separation step is recycled back into the apparatusused in the second separation step; and part of the raffinate streamfrom the apparatus used in the second separation step is recycled backinto the apparatus used in the second separation step; and part of theextract stream from the apparatus used in the third separation step isrecycled back into the apparatus used in the third separation step; andpart of the raffinate stream from the apparatus used in the thirdseparation step is recycled back into the apparatus used in the thirdseparation step.

This recycle involves feeding part of the extract or raffinate streamout of the chromatography apparatus used in the first, second or thirdseparation step back into the apparatus used in that step, typicallyinto an adjacent column. This adjacent column is the adjacent columnwhich is downstream with respect to the flow of eluent in the system.

When two or three separation steps are carried out simultaneously in asingle chromatography in two or three zones respectively, this recycleinvolves recycling the particular extract or raffinate stream removedfrom a zone back into the same zone.

The rate at which liquid collected via the extract or raffinate streamin a particular separation step is recycled back into a chromatographyapparatus or zone used in that separation step is the rate at whichliquid collected via that stream is fed back into the apparatus used inthat step, typically into an adjacent column, i.e. the downstream columnwith respect to the flow of eluent in the system.

This can be seen with reference to a preferred embodiment in FIG. 5. Therate of recycle of extract in the first separation step is the rate atwhich extract collected from the bottom of column 2 of thechromatographic apparatus used in the first separation step is fed intothe top of column 3 of the chromatographic apparatus used in the firstseparation step, i.e. the flow rate of liquid into the top of column 3of the chromatographic apparatus used in the first separation step.

The rate of recycle of extract in the second separation step is the rateat which extract collected at the bottom of column 10 of thechromatographic apparatus used in the second separation step is fed intothe top of column 11 of the chromatographic apparatus used in the secondseparation step, i.e. the flow rate of liquid into the top of column 11of the chromatographic apparatus used in the second separation step.

The rate of recycle of extract in the third separation step is the rateat which extract collected at the bottom of column 19 of thechromatographic apparatus used in the second separation step is fed intothe top of column 19 of the chromatographic apparatus used in the secondseparation step, i.e. the flow rate of liquid into the top of column 19of the chromatographic apparatus used in the second separation step.

Recycle of the extract and/or raffinate streams in the first, secondand/or third separation steps is typically effected by feeding theliquid collected via that stream in that separation step into acontainer, and then pumping an amount of that liquid from the containerback into the apparatus or zone used in that separation step, typicallyinto an adjacent column. In this case, the rate of recycle of liquidcollected via a particular extract or raffinate stream in the firstand/or second separation steps, typically back into an adjacent column,is the rate at which liquid is pumped out of the container back into thechromatography apparatus or zone, typically into an adjacent column.

As the skilled person will appreciate, the amount of liquid beingintroduced into a chromatography apparatus via the eluent and feedstockstreams is balanced with the amount of liquid removed from theapparatus, and recycled back into the apparatus.

Thus, with reference to FIG. 5, for the extract stream, the flow rate ofeluent (desorbent) into the chromatographic apparatus(es) used in thesecond and third separation steps (D) is equal to the rate at whichliquid collected via the extract stream in that separation stepaccumulates in a container (E2 and E3) added to the rate at whichextract is recycled back into the chromatographic apparatus used in thatparticular separation step (D-E2 and D-E3).

For the raffinate stream from a separation step, the rate at whichextract is recycled back into the chromatographic apparatus used in thatparticular separation step (D-E1 and D-E2) added to the rate at whichfeedstock is introduced into the chromatographic apparatus used in thatparticular separation step (F and R1) is equal to the rate at whichliquid collected via the raffinate stream in that particular separationstep accumulates in a container (R1 and R2) added to the rate at whichraffinate is recycled back into the chromatographic apparatus used inthat particular separation step (D+F-E1-R1 and D+R1-E2-R2).

The rate at which liquid collected from a particular extract orraffinate stream from a chromatography apparatus or zone accumulates ina container can also be thought of as the net rate of removal of thatextract or raffinate stream from that chromatography apparatus.

Typically, the rate at which liquid collected via one or both of theextract and raffinate streams in the second separation step is recycledback into the apparatus used in that separation step is adjusted suchthat saturated and/or monounsaturated fatty acids present in the feedmixture are removed in the first separation step, and the PUFA productis separated from different components of the feed mixture in steps (ii)and (iii); and/or wherein the rate at which liquid collected via one orboth of the extract and raffinate streams in the third separation stepis recycled back into the apparatus used in that separation step isadjusted such that saturated and/or monounsaturated fatty acids presentin the feed mixture are removed in the first separation step, and thePUFA product is separated from different components of the feed mixturein steps (ii) and (iii).

Preferably, the rate at which liquid collected via one or both of theextract and raffinate streams in the second separation step is recycledback into the apparatus used in that separation step is adjusted suchthat saturated and/or monounsaturated fatty acids present in the feedmixture are removed in the first separation step, and the PUFA productis separated from different components of the feed mixture in steps (ii)and (iii); and wherein the rate at which liquid collected via one orboth of the extract and raffinate streams in the third separation stepis recycled back into the apparatus used in that separation step isadjusted such that saturated and/or monounsaturated fatty acids presentin the feed mixture are removed in the first separation step, and thePUFA product is separated from different components of the feed mixturein steps (ii) and (iii).

When the first separation step comprises purifying the feed mixture in asimulated or actual moving bed chromatography apparatus, the rate atwhich liquid collected via one or both of the extract and raffinatestreams in the first separation step is recycled back into the apparatusused in that separation step is typically adjusted such that saturatedand/or monounsaturated fatty acids present in the feed mixture areremoved in the first separation step, and the PUFA product is separatedfrom different components of the feed mixture in steps (ii) and (iii).

Typically, the rate at which liquid collected via the extract stream inthe second separation step is recycled back into the chromatographyapparatus used in the second separation step differs from the rate atwhich liquid collected via the extract stream in the third separationstep is recycled back into the chromatography apparatus used in thethird separation step; and/or the rate at which liquid collected via theraffinate stream in the second separation step is recycled back into thechromatography apparatus used in the second separation step differs fromthe rate at which liquid collected via the raffinate stream in the thirdseparation step is recycled back into the chromatography apparatus usedin the third separation step.

When the first separation step comprises purifying the feed mixture in asimulated or actual moving bed chromatography apparatus, the rate atwhich liquid collected via the extract stream in the first separationstep is recycled back into the chromatography apparatus used in thefirst separation step typically differs from the rate at which liquidcollected via the extract stream in the second separation step isrecycled back into the chromatography apparatus used in the secondseparation step; and/or the rate at which liquid collected via theraffinate stream in the first separation step is recycled back into thechromatography apparatus used in the first separation step typicallydiffers from the rate at which liquid collected via the raffinate streamin the second separation step is recycled back into the chromatographyapparatus used in the second separation step

Varying the rate at which liquid collected via the extract and/orraffinate streams in the first, second and/or third separation steps isrecycled back into the apparatus used in that particular separation stephas the effect of varying the amount of more polar and less polarcomponents present in the extract and raffinate streams. Thus, forexample, a lower extract recycle rate results in fewer of the less polarcomponents in that separation step being carried through to theraffinate stream. A higher extract recycle rate results in more of theless polar components in that separation step being carried through tothe raffinate stream.

This can be seen, for example, in the specific embodiment of theinvention shown in FIG. 5. The rate at which liquid collected via theextract stream in the second separation step is recycled back into thechromatographic apparatus used in that separation step (D-E2) willaffect to what extent any of component A is carried through to theraffinate stream in the second separation step (R2).

Typically, the rate at which liquid collected via the extract stream inthe second separation step is recycled back into the chromatographicapparatus used in the second separation step is faster than the rate atwhich liquid collected via the extract stream in the third separationstep is recycled back into the chromatographic apparatus used in thethird separation step. Preferably, a raffinate stream containing thePUFA product together with more polar components is collected from thesecond separation step and purified in the third separation step, andthe rate at which liquid collected via the extract stream in the secondseparation step is recycled back into the chromatographic apparatus usedin the second separation step is faster than the rate at which liquidcollected via the extract stream in the third separation step isrecycled back into the chromatographic apparatus used in the thirdseparation step.

Alternatively, the rate at which liquid collected via the raffinatestream in the second separation step is recycled back into thechromatographic apparatus used in the second separation step is fasterthan the rate at which liquid collected via the raffinate stream in thethird separation step is recycled back into the chromatographicapparatus used in the third separation step. Preferably, an extractstream containing the PUFA product together with less polar componentsis collected from the second separation step and purified in the thirdseparation step, and the rate at which liquid collected via theraffinate stream in the second separation step is recycled back into thechromatographic apparatus used in the second separation step is fasterthan the rate at which liquid collected via the raffinate stream in thethird separation step is recycled back into the chromatographicapparatus used in the third separation step.

Where recycle rates are adjusted such that saturated and/ormonounsaturated fatty acids present in the feed mixture are removed inthe first separation step, and the PUFA product is separated fromdifferent components of the feed mixture in steps (ii) and (iii), thewater:organic solvent ratio of the eluents used in the separation stepswhere the recycle rates differ may be the same or different.

The eluent used in the process of the present invention is an aqueousorganic solvent.

The aqueous organic solvent typically comprises water and one or morealcohols, ethers, esters, ketones or nitriles, or mixtures thereof.

Alcohol solvents are well known to the person skilled in the art.Alcohols are typically short chain alcohols. Alcohols typically are offormula ROH, wherein R is a straight or branched C₁-C₆ alkyl group. TheC₁-C₆ alkyl group is preferably unsubstituted. Examples of alcoholsinclude methanol, ethanol, n-propanol, propanol, n-butanol, i-butanol,s-butanol and t-butanol. Methanol and ethanol are preferred. Methanol ismore preferred.

Ether solvents are well known to the person skilled in the art. Ethersare typically short chain ethers. Ethers typically are of formulaR—O—R′, wherein R and R′ are the same or different and represent astraight or branched C₁-C₆ alkyl group. The C₁-C₆ alkyl group ispreferably unsubstituted. Preferred ethers include diethylether,diisopropylether, and methyl t-butyl ether (MTBE).

Ester solvents are well known to the person skilled in the art. Estersare typically short chain esters. Esters typically are of formulaR—(C═O)O—R′, wherein R and R′ are the same or different and represent astraight or branched C₁-C₆ alkyl group. Preferred esters includemethylacetate and ethylacetate.

Ketone solvents are well known to the person skilled in the art. Ketonesare typically short chain ketones. Ketones typically are of formulaR—(C═O)—R′, wherein R and R′ are the same or different and represent astraight or branched C₁-C₆ alkyl group. The C₁-C₆ alkyl group ispreferably unsubstituted. Preferred ketones include acetone,methylethylketone and methyl isobutyl ketone (MIBK).

Nitrile solvents are well known to the person skilled in the art.Nitriles are typically short chain nitriles. Nitriles typically are offormula R—CN, wherein R represents a straight or branched C₁-C₆ alkylgroup. The C₁-C₆ alkyl group is preferably unsubstituted. Preferrednitriles include acetonitrile.

Typically, the aqueous organic solvent is aqueous alcohol or aqueousacetonitrile.

The aqueous organic solvent is preferably aqueous methanol or aqueousacetonitrile. Aqueous methanol is more preferred.

Typically, the eluent is not in a supercritical state. Typically, theeluent is a liquid.

Typically, the average water:organic solvent ratio, for examplewater:methanol ratio, of the eluent in the entire apparatus is from0.1:99.9 to 9:91 wt %, preferably from 0.25:99.75 to 7:93 wt %, morepreferably from 0.5:99.5 to 6:94 wt %.

When the aqueous organic solvent is aqueous acetonitrile, the eluenttypically contains up to 30 wt % water, remainder acetonitrile.Preferably, the eluent contains from 5 to 25 wt % water, remainderacetonitrile. More preferably, the eluent contains from 10 to 20 wt %water, remainder acetonitrile. Even more preferably, the eluent containsfrom 15 to 25 wt % water, remainder acetonitrile.

Typically, the water:organic solvent ratio used in each separation stepis adjusted such that saturated and/or monounsaturated fatty acidspresent in the feed mixture are removed in the first separation step;and the PUFA product is separated from different components of the feedmixture in steps (ii) and (iii).

Typically, the aqueous organic solvent eluent used in two or more of theseparation steps has a different water:organic solvent ratio. In oneembodiment, the water:organic solvent ratio used in each separation stephas a different water:organic solvent ratio.

The eluting power of the eluent used in two or more of the separationsteps is typically different. Depending on the choice of organicsolvent, they may be more powerful desorbers than water. Alternatively,they may be less powerful desorbers than water. Acetonitrile andalcohols, for example, are more powerful desorbers than water.

In a preferred embodiment, the aqueous organic solvent eluent used inthe second and third separation steps has the same water:organic solventratio, and the aqueous organic solvent eluent used in the firstseparation step has a different water:organic solvent ratio from theorganic solvent eluent used in the second and third separation steps.

In this preferred embodiment, the eluting power of the eluent used inthe second and third separation steps is the same; and/or the elutingpower of the eluent used in the first separation step is greater thanthat of the eluent used in the second separation step. Preferably inthis embodiment, the eluting power of the eluent used in the second andthird separation steps is the same; and the eluting power of the eluentused in the first separation step is greater than that of the eluentused in the second and third separation steps. In this embodiment, whenthe aqueous organic solvent is aqueous alcohol or acetonitrile, theamount of alcohol or acetonitrile in the eluent used in the second andthird separation steps is typically the same, and the amount of alcoholor acetonitrile in the eluent used in the first separation step istypically greater than the amount of alcohol or acetonitrile in theeluent used in the second and third separation steps. Thus, in thisembodiment, the water:organic solvent ratio of the eluent in the secondand third separation steps is typically the same, and the water:organicsolvent ratio of the eluent in the first separation step is typicallylower than the water:organic solvent ratio of the eluent in the secondand third separation steps.

In this preferred embodiment, the water:organic solvent ratio of theeluent in the first separation step is typically from 0:100 to 5:95 wt%, preferably from 0.1:99.9 to 2.5:97.5 wt %, more preferably from0.1:99.9 to 2:98 wt %, even more preferably from 0.1:99.9 to 1:99 wt %,even more preferably from 0.25:99.75 to 0.75:99.25 wt %, and mostpreferably about 0.5:99.5. In this preferred embodiment, thewater:organic solvent ratio of the eluent in the second and thirdseparation steps is typically from 5:95 to 11:89 wt %, preferably 6:94to 10:90 wt %, more preferably from 7:93 to 9:91 wt %, even morepreferably from 7.5:92.5 to 8.5:91.5 wt %, and most preferably about8:92 wt %.

In this preferred embodiment, the water:organic solvent ratio of theeluent used in the first separation step is preferably from 0.1:99.9 to1:99 wt %, and the water:organic solvent ratio of the eluent used in thesecond and third separation steps is preferably from 7:93 to 9:91 wt %.

In an alternative embodiment, the aqueous organic solvent eluent used ineach separation step has a different water:organic solvent ratio.

In this alternative embodiment, the eluting power of the eluent used inthe first separation step is greater than that of the eluent used in thesecond separation step; and/or the eluting power of the eluent used inthe second separation step is greater than that of the eluent used inthe third separation step. Preferably, a raffinate stream containing thePUFA product together with more polar components is collected from thesecond separation step and purified in the third separation step and theeluting power of the eluent used in the second separation step isgreater than that of the eluent used in the third separation step.Alternatively, an extract stream containing the PUFA product togetherwith less polar components is collected from the second separation stepand purified in the third separation step and the eluting power of theeluent used in the second separation step is lower than that of theeluent used in the third separation step.

In practice this is achieved by varying the relative amounts of waterand organic solvent used in each separation step. In this embodiment,when the aqueous organic solvent is aqueous alcohol or acetonitrile, theamount of alcohol or acetonitrile in the eluent used in the firstseparation step is typically greater than the amount of alcohol oracetonitrile in the eluent used in the second separation step; and/orthe amount of alcohol or acetonitrile in the eluent used in the secondseparation step is typically greater than the amount of alcohol oracetonitrile in the eluent used in the third separation step. Thus, inthis embodiment, the water:organic solvent ratio of the eluent in thefirst separation step is typically lower than the water:organic solventratio of the eluent in the second separation step; and/or thewater:organic solvent ratio of the eluent in the second separation stepis typically lower than the water:organic solvent ratio of the eluent inthe third separation step.

It will be appreciated that the ratios of water and organic solvent ineach separation step referred to above are average ratios within thetotality of the chromatographic apparatus.

Typically, the water:organic solvent ratio of the eluent in eachseparation step is controlled by introducing water and/or organicsolvent into one or more columns in the chromatographic apparatuses usedin the separation steps. Thus, for example, to achieve a lowerwater:organic solvent ratio in the first separations step than in thesecond and third separation steps, water is typically introduced moreslowly into the chromatographic apparatus used in the first separationstep than in the second and third separation steps.

In some embodiments, essentially pure organic solvent and essentiallypure water may be introduced at different points in the chromatographicapparatus used in each separation step. The relative flow rates of thesetwo streams will determine the overall solvent profile in thechromatographic apparatus. In other embodiments, different organicsolvent/water mixtures may be introduced at different points in eachchromatographic apparatus used in each separation step. That willinvolve introducing two or more different organic solvent/water mixturesinto the chromatographic apparatus used in a particular separation step,each organic solvent/water mixture having a different organicsolvent:water ratio. The relative flow rates and relative concentrationsof the organic solvent/water mixtures in this embodiment will determinethe overall solvent profile in the chromatographic apparatus used inthat separation step.

Preferably, the aqueous organic solvent eluent used in the second andthird separation steps has the same water:organic solvent ratio, and theaqueous organic solvent eluent used in the first separation step has adifferent water:organic solvent ratio from the organic solvent eluentused in the second and third separation steps; and the rate at whichliquid collected via the extract stream in the second separation step isrecycled back into the chromatography apparatus used in the secondseparation step differs from the rate at which liquid collected via theextract stream in the third separation step is recycled back into thechromatography apparatus used in the third separation step.

More preferably, the water:organic solvent ratio of the eluent in thesecond and third separation steps is the same, and the water:organicsolvent ratio of the eluent in the first separation step is lower thanthe water:organic solvent ratio of the eluent in the second and thirdseparation steps; and the rate at which liquid collected via the extractstream in the second separation step is recycled back into thechromatographic apparatus used in the second separation step is fasterthan the rate at which liquid collected via the extract stream in thethird separation step is recycled back into the chromatographicapparatus used in the third separation step.

Even more preferably,

-   the first separation step comprises purifying the feed mixture in a    simulated or actual moving bed chromatography apparatus;-   the second and third separation steps are carried out simultaneously    in a single simulated or actual moving bed chromatography apparatus    having a plurality of linked chromatography columns containing, as    eluent, an aqueous organic solvent, the second and third separation    steps being carried out in first and second zones respectively,    wherein each zone is as defined herein, and wherein the first    separation step is carried out in a separate simulated or actual    moving bed chromatography apparatus;-   the first intermediate product is collected as the raffinate stream    in the first separation step, the second intermediate product is    collected as the raffinate stream in the second separation step, and    the PUFA product is collected as the extract stream in the third    separation step;-   the second intermediate product raffinate stream containing the PUFA    product together with more polar components is collected from a    column in the first zone and introduced into a nonadjacent column in    the second zone;-   the aqueous organic solvent eluent used in the second and third    separation steps has the same water:organic solvent ratio, and the    water:organic solvent ratio of the eluent used in the first    separation step is lower than the water:organic solvent ratio of the    eluent used in the second and third separation steps; and-   the rate at which liquid collected via the extract stream in the    second separation step is recycled back into the chromatographic    apparatus used in the second separation step is faster than the rate    at which liquid collected via the extract stream in the third    separation step is recycled back into the chromatographic apparatus    used in the third separation step.

It is preferred that the first separation step comprises purifying thefeed mixture in a simulated or actual moving bed chromatographyapparatus; and the second and third separation steps are carried outsimultaneously in a single simulated or actual moving bed chromatographyapparatus having a plurality of linked chromatography columnscontaining, as eluent, an aqueous organic solvent, the second and thirdseparation steps being carried out in first and second zonesrespectively, wherein each zone is as defined herein, and wherein thefirst separation step is carried out in a separate simulated or actualmoving bed chromatography apparatus. A preferred embodiment of this isillustrated in FIG. 3.

A feed mixture F comprising the PUFA product (B) and more polar (C) andless polar (A′) and (A) components is purified in the first separationstep. In the first separation step, the least polar components (e.g.saturates and/or monounsaturates) (A′) are removed as extract stream E1.The PUFA product (B), more polar components (C) and less polar (but morepolar than (A′)) components (A) are collected as raffinate stream R1.Raffinate stream R1 is the intermediate product which is then purifiedin the second separation step.

In the second separation step, the less polar components (A) are removedas extract stream E2. The PUFA product (B) and more polar components (C)are collected as raffinate stream R2. Raffinate stream R2 is theintermediate product which is then purified in the third separationstep.

In the third separation step, more polar components (C) are removed asraffinate stream R3. The PUFA product (B) is collected as extract streamE3. The second and third separation steps take place in two zones in asingle SMB chromatographic apparatus.

This embodiment is illustrated in more detail in FIG. 4. FIG. 4 isidentical to FIG. 2, except that the points of introduction of theaqueous organic solvent desorbent (D) into each chromatographicapparatus are shown.

Typical solvents for use in this most preferred embodiment are aqueousalcohols or aqueous acetonitrile, preferably aqueous methanol.

Typically in this preferred embodiment, the aqueous organic solventeluent used in the second and third separation steps has the samewater:organic solvent ratio, and the aqueous organic solvent eluent usedin the first separation step has a different water:organic solvent ratiofrom the organic solvent eluent used in the second and third separationsteps; and the rate at which liquid collected via the extract stream inthe second separation step is recycled back into the chromatographyapparatus used in the second separation step differs from the rate atwhich liquid collected via the extract stream in the third separationstep is recycled back into the chromatography apparatus used in thethird separation step.

Preferably in this preferred embodiment, the water:organic solvent ratioof the eluent in the second and third separation steps is the same, andthe water:organic solvent ratio of the eluent in the first separationstep is lower than the water:organic solvent ratio of the eluent in thesecond and third separation steps; and the rate at which liquidcollected via the extract stream in the second separation step isrecycled back into the chromatographic apparatus used in the secondseparation step is faster than the rate at which liquid collected viathe extract stream in the third separation step is recycled back intothe chromatographic apparatus used in the third separation step.

In this preferred embodiment the first raffinate stream in the firstseparation step is typically removed downstream of the point ofintroduction of the feed mixture into the chromatographic apparatus usedin the first separation step, with respect to the flow of eluent.

In this particularly preferred embodiment, the first extract stream inthe first separation step is typically removed upstream of the point ofintroduction of the feed mixture into the chromatographic apparatus usedin the first separation step, with respect to the flow of eluent.

In this particularly preferred embodiment, the second raffinate streamin the second separation step is typically removed downstream of thepoint of introduction of the first intermediate product into thechromatographic apparatus used in the second separation step, withrespect to the flow of eluent.

In this particularly preferred embodiment, the second extract stream inthe second separation step is typically collected upstream of the pointof introduction of the first intermediate product into thechromatographic apparatus used in the second separation step, withrespect to the flow of eluent.

In this particularly preferred embodiment, the third raffinate stream inthe third separation step is typically removed downstream of the pointof introduction of the second intermediate product into thechromatographic apparatus used in the third separation step, withrespect to the flow of eluent.

In this particularly preferred embodiment, the third extract stream inthe third separation step is typically collected upstream of the pointof introduction of the second intermediate product into thechromatographic apparatus used in the third separation step, withrespect to the flow of eluent.

Typically in this preferred embodiment, the aqueous organic solvent isintroduced into the chromatographic apparatus used in the firstseparation step upstream of the point of removal of the first extractstream, with respect to the flow of eluent.

Typically in this preferred embodiment, the aqueous organic solvent isintroduced into the chromatographic apparatus used in the secondseparation step upstream of the point of removal of the second extractstream, with respect to the flow of eluent.

Typically in this preferred embodiment, the aqueous organic solvent isintroduced into the chromatographic apparatus used in the thirdseparation step upstream of the point of removal of the third extractstream, with respect to the flow of eluent.

A more preferred embodiment of the invention illustrated in FIGS. 3 and4 is shown in FIG. 5. This illustrates the number of columns used ineach separation step, and shows typical points of introduction of feedmixtures and eluents, and typical points of removal of extract andraffinate streams.

Thus, in this more preferred embodiment, the SMB chromatographyapparatus used in the first separation step consists of eightchromatographic columns, 1 to 8. The SMB chromatography apparatus usedin the second separation step consists of eight chromatographic columns,9 to 16. The SMB chromatography apparatus used in the third separationstep consists of seven chromatographic columns, 17 to 23.

In each apparatus the columns are typically arranged in series so that(in the case of the first separation step) the bottom of column 1 islinked to the top of column 2, the bottom of column 2 is linked to thetop of column . . . 3 etc . . . and the bottom of column 8 is linked tothe top of column 1. These linkages may optionally be via a holdingcontainer, with a recycle stream into the next column. The flow ofeluent through the system is from column 1 to column 2 to column 3 etc.The effective flow of adsorbent through the system is from column 8 tocolumn 7 to column 6 etc.

In this more preferred embodiment, a feed mixture F comprising the PUFAproduct (B) and more polar (C) and less polar (A′) and (A) components isintroduced into the top of column 5 in the chromatographic apparatusused in the first separation step. Aqueous organic solvent desorbent isintroduced into the top of column 1 of the chromatographic apparatusused in the first separation step. In the first separation step, theleast polar components (e.g. saturates and/or monounsaturates) (A′) areremoved as extract stream E1 from the bottom of column 2. The PUFAproduct (B), more polar components (C) and less polar (but more polarthan (A′)) components (A) are collected as raffinate stream R1 from thebottom of column 6.

Raffinate stream R1 is the first intermediate product which is thenpurified in the second separation step, by being introduced into thechromatographic apparatus used in the second separation step at the topof column 13. Aqueous organic solvent desorbent is introduced into thetop of column D in the chromatographic apparatus used in the secondseparation step.

In the second separation step, the less polar components (A) are removedas extract stream E2 at the bottom of column 10. The PUFA product (B)and more polar components (C) are collected as raffinate stream R2 atthe bottom of column 14. Raffinate stream R2 is the intermediate productwhich is then purified in the third separation step, by being introducedinto the chromatographic apparatus used in the second separation step atthe top of column 21.

In the third separation step, more polar components (C) are removed asraffinate stream R3 at the bottom of column 22. The PUFA product (B) iscollected as extract stream E3 at the bottom of column 18. The secondand third separation steps take place in two zones in a single SMBchromatographic apparatus.

In this more preferred embodiment, aqueous organic solvent is typicallyintroduced into the top of column 1 of the chromatographic apparatusused in the first separation step.

In this more preferred embodiment, aqueous organic solvent is typicallyintroduced into the top of column 9 of the chromatographic apparatusused in the second separation step.

In this more preferred embodiment, aqueous organic solvent is typicallyintroduced into the top of column 17 of the chromatographic apparatusused in the third separation step.

In this more preferred embodiment, the feed stream is typicallyintroduced into the top of column 5 of the chromatographic apparatusused in the first separation step.

In this more preferred embodiment, a first raffinate stream is typicallycollected as the first intermediate product from the bottom of column 6of the chromatographic apparatus used in the first separation step. Thisfirst intermediate product is then purified in the second separationstep and is typically introduced into the top of column 13 of thechromatographic apparatus used in the second separation step. The firstraffinate stream may optionally be collected in a container before beingpurified in the second separation step.

In this more preferred embodiment, a first extract stream is typicallyremoved from the bottom of column 2 of the chromatographic apparatusused in the first separation step. The first extract stream mayoptionally be collected in a container and reintroduced into the top ofcolumn 3 of the chromatographic apparatus used in the first separationstep.

In this more preferred embodiment, a second raffinate stream istypically collected as the second intermediate product from the bottomof column 14 of the chromatographic apparatus used in the secondseparation step. This second intermediate product is then purified inthe third separation step and is typically introduced into the top ofcolumn 21 of the chromatographic apparatus used in the third separationstep. The second raffinate stream may optionally be collected in acontainer before being purified in the second separation step.

In this more preferred embodiment, a second extract stream is typicallyremoved from the bottom of column 10 of the chromatographic apparatusused in the second separation step.

In this more preferred embodiment, a third extract stream is typicallycollected from the bottom of column 18 of the chromatographic apparatusused in the third separation step. This third extract stream typicallycontains the purified PUFA product. The third extract stream mayoptionally be collected in a container and reintroduced into the top ofcolumn 19 of the chromatographic apparatus used in the third separationstep.

In this more preferred embodiment, a third raffinate stream is typicallyremoved from the bottom of column 22 of the chromatographic apparatusused in the third separation step.

Typically in this more preferred embodiment, the aqueous organic solventeluent used in the second and third separation steps has the samewater:organic solvent ratio, and the aqueous organic solvent eluent usedin the first separation step has a different water:organic solvent ratiofrom the organic solvent eluent used in the second and third separationsteps; and the rate at which liquid collected via the extract stream inthe second separation step is recycled back into the chromatographyapparatus used in the second separation step differs from the rate atwhich liquid collected via the extract stream in the third separationstep is recycled back into the chromatography apparatus used in thethird separation step.

Preferably in this more preferred embodiment, the water:organic solventratio of the eluent in the second and third separation steps is thesame, and the water:organic solvent ratio of the eluent in the firstseparation step is lower than the water:organic solvent ratio of theeluent in the second and third separation steps; and the rate at whichliquid collected via the extract stream in the second separation step isrecycled back into the chromatographic apparatus used in the secondseparation step is faster than the rate at which liquid collected viathe extract stream in the third separation step is recycled back intothe chromatographic apparatus used in the third separation step.

In this more preferred embodiment, the water:organic solvent ratio ofthe eluent used in the second and third separation steps is the same andis from 7:93 to 9:91 wt %, and the water:organic solvent ratio of theeluent in the first separation step is from 0.1:99.9 to 1:99 wt %.

Although these preferred and more preferred embodiments are shown as forFIG. 2C discussed above, they may also be carried out with apparatusesconfigured such that:

-   the first separation step comprises purifying the feed mixture in a    simulated or actual moving bed chromatography apparatus, and the    first, second and third separation steps are carried out    simultaneously in a single simulated or actual moving bed    chromatography apparatus having a plurality of linked chromatography    columns containing, as eluent, an aqueous organic solvent, the    first, second and third separation steps being carried out in first,    second and third zones respectively, wherein each zone is as defined    herein; or-   the first separation step comprises purifying the feed mixture in a    simulated or actual moving bed chromatography apparatus, and the    second and third separation steps are carried out simultaneously in    a single simulated or actual moving bed chromatography apparatus    having a plurality of linked chromatography columns containing, as    eluent, an aqueous organic solvent, the second and third separation    steps being carried out in first and second zones respectively,    wherein each zone is as defined herein, and wherein the first    separation step is carried out in a separate simulated or actual    moving bed chromatography apparatus; or-   the first separation step comprises purifying the feed mixture in a    simulated or actual moving bed chromatography apparatus, and (a) the    first, second and third separation steps are carried out    sequentially on the same chromatography apparatus, first and second    intermediate products being recovered between the first and second,    and second and third separation steps respectively, and the process    conditions in the chromatography apparatus being adjusted between    the first and second, and second and third separation steps such    that saturated and/or monounsaturated fatty acids present in the    feed mixture are removed in the first separation step, and the PUFA    product is separated from different components of the feed mixture    in steps (ii) and (iii); or-   (b) the second separation step is carried out using a different    chromatographic apparatus to that used in the first separation step,    and/or the third separation step is carried out using a different    chromatographic apparatus to that used in the second separation    step; or-   the first separation step comprises purifying the feed mixture in a    stationary bed chromatography apparatus, and the second and third    separation steps are carried out simultaneously in a single    simulated or actual moving bed chromatography apparatus having a    plurality of linked chromatography columns containing, as eluent, an    aqueous organic solvent, the second and third separation steps being    carried out in first and second zones respectively, wherein each    zone is as defined herein; or-   the first separation step comprises purifying the feed mixture in a    stationary bed chromatography apparatus, and the second and third    separation steps are carried out sequentially on the same    chromatography apparatus, the second intermediate product being    recovered between the second and third separation steps and the    process conditions in the chromatography apparatus being adjusted    between the second and third separation steps such that the PUFA    product is separated from different components of the feed mixture    in steps (ii) and (iii); or-   the first separation step comprises purifying the feed mixture in a    stationary bed chromatography apparatus, and the second and third    separation steps are carried out on separate chromatography    apparatuses respectively, the intermediate product obtained from the    second separation step being introduced into the chromatography    apparatus used in the third separation step.

The process of the invention allows much higher purities of PUFA productto be achieved than have been possible with conventional chromatographictechniques. PUFA products produced by the process of the invention alsohave particularly advantageous impurity profiles, which are quitedifferent from those observed in oils prepared by known techniques. Thepresent invention therefore also relates to compositions comprising aPUFA product, for example one obtainable by the process of the presentinvention.

In practice, the process of the present invention will generally becontrolled by a computer. The present invention therefore also providesa computer program for controlling a chromatographic apparatus asdefined herein, the computer program containing code means that whenexecuted instruct the apparatus to carry out the process of theinvention.

The following Examples illustrate the invention.

EXAMPLES Example 1

A fish oil derived feedstock (55 weight % EPA EE, 5 weight % DHA EE) isfractionated using an actual moving bed chromatography system usingbonded C18 silica gel as stationary phase and aqueous methanol as eluentaccording to the system schematically illustrated in FIG. 5. A GC traceof the feed mixture is shown as FIG. 7.

In a first separation step, the feed mixture was passed through an SMBapparatus having 8 columns 1 to 8 (diameter: 152 mm, length: 813 mm)connected in series as shown FIG. 5. Process conditions were adjusted toremove saturated and monounsaturated components from the feed mixture asthe extract stream. A 0.5:99.5 wt % water:methanol eluent was used. Theraffinate stream was retained as the first intermediate product. A GCtrace of the first intermediate product is shown as FIG. 8.

The first intermediate product was passed through an SMB apparatushaving two zones with eight columns, columns 9 to 16, in the first zoneand seven columns, columns 17 to 23, in the second zone. An 8:92 wt %water:methanol eluent was used in both first and second zones, i.e. inboth the second and third separation steps. The process conditions inthe first zone were adjusted to purify EPA from the slower runningcomponents such as DHA, which were removed as the extract stream. Theraffinate stream was retained as the second intermediate product. A GCtrace of the second intermediate product is shown as FIG. 9.

The second intermediate product was then introduced into the second zoneand separated from the faster running components, which were removed asa raffinate stream. High purity EPA was collected as the extract streamfrom the second zone. A GC trace of the EPA PUFA product is shown asFIG. 10.

EPA was produced with a final purity of greater than 97%.

It can be seen that for the three separation steps taken together, theoverall rate of accumulation of extract (E1+E2+E3) 3876 ml/min.

The process conditions for each separation step are as follows:

First Separation Step

-   Feedstock feed rate: 94 ml/min-   Desorbent feed rate: 6250 ml/min-   Extract accumulation rate: 1250 ml/min-   Extract recycle rate: 5000 ml/min-   Raffinate accumulation rate: 1688 ml/min-   Cycle time: 600 secs    Second Separation Step-   First intermediate product feed rate: 40 ml/min-   Desorbent feed rate: 6313 ml/min-   Extract accumulation rate: 1188 ml/min-   Extract recycle rate: 5125 ml/min-   Raffinate accumulation rate: 1625 ml/min-   Cycle time: 1200 secs    Third Separation Step-   Second intermediate product feed rate: 40 ml/min-   Desorbent feed rate: 6189 ml/min-   Extract accumulation rate: 1438 ml/min-   Extract recycle rate: 4750 ml/min-   Raffinate accumulation rate: 1438 ml/min-   Cycle time: 1080 secs

Comparative Example 1

An experiment was carried out to produce a PUFA product containinggreater than 97% EPA from the same feed mixture as was used inExample 1. However, instead of using a three step separation process inaccordance with the present invention, only two separation steps wereused. Thus, the process was carried out in accordance with the processdisclosed in PCT/GB10/002339, and as illustrated in FIG. 6.

A single chromatographic apparatus having two zones was used as shown inFIG. 6. The first zone contains 8 columns (diameter: 24″, length: 32″)and the second zone 7 columns (diameter: 24″, length: 32″). Processconditions were adjusted to separate the EPA PUFA product from lesspolar components of the feed mixture in the first zone, and more polarcomponents of the feed mixture in the second zone. An 8:92 wt %water:methanol eluent was used in both zones.

EPA was produced with a final purity of greater than 97%.

It can be seen that for the two separation steps taken together, theoverall rate of accumulation of extract (E1+E2) was 10571 ml/min. Thus,it can be seen that a much higher volume of aqueous organic solvent isrequired to recover the PUFA product compared with the three stepprocess of the invention.

The process conditions for the separation steps are as follows:

First Separation Step

-   Feed mixture feed rate: 34 ml/min-   Desorbent feed rate: 14438 ml/min-   Extract accumulation rate: 9313 ml/min-   Extract recycle rate: 5125 ml/min-   Raffinate accumulation rate: 1688 ml/min-   Cycle time: 1200 secs    Third Separation Step-   Intermediate product feed rate: 40 ml/min-   Desorbent feed rate: 6189 ml/min-   Extract accumulation rate: 1438 ml/min-   Extract recycle rate: 4750 ml/min-   Raffinate accumulation rate: 1438 ml/min-   Cycle time: 1080 secs

The invention claimed is:
 1. A chromatographic separation process forrecovering a polyunsaturated fatty acid (PUFA) product from a feedmixture which is a fish oil or which is derived from fish oil, whichprocess comprises the steps of: (i) purifying the feed mixture in achromatographic first separation step, to obtain a first intermediateproduct; and (ii) purifying the first intermediate product obtained in(i) in a simulated or actual moving bed chromatographic secondseparation step, to obtain a second intermediate product; and (iii)purifying the second intermediate product obtained in (ii) in achromatographic third separation step using a different chromatographicapparatus to that used in the second separation step, to obtain the PUFAproduct; wherein an aqueous organic solvent is used as eluent in eachseparation step; saturated and/or monounsaturated fatty acids present inthe feed mixture are removed in the first separation step; the PUFAproduct is separated from different components of the feed mixture insteps (ii) and (iii); and the PUFA product obtained in the thirdseparation step contains EPA or an EPA derivative in an amount greaterthan 90 wt %.
 2. The process according to claim 1, wherein steps (i),(ii) and (iii) are carried out on separate chromatographic apparatuses.3. The process according to claim 1, wherein the first intermediateproduct is recovered between the first and second separation steps. 4.The process according to claim 1, wherein the second intermediateproduct is recovered between the second and third separation steps. 5.The process according to claim 1, wherein the aqueous organic solventeluent is partly or totally removed from the intermediate productobtained in the first and/or second separation step before theintermediate product is purified further in the next separation step. 6.The process according to claim 1, wherein the eluent is not sharedbetween the separate chromatographic apparatuses.
 7. The processaccording to claim 1, wherein the first intermediate product obtained inthe first separation step is enriched in the PUFA product compared tothe feed mixture; and the second intermediate product obtained in thesecond separation step is enriched in the PUFA product compared to thefirst intermediate product; and/or wherein in the first step the PUFAproduct is separated from components of the feed mixture which are lesspolar than the PUFA product, in the second step the PUFA product isseparated from components of the feed mixture which are less polar thanthe PUFA product but more polar than the components separated in thefirst separation step, and in the third separation step the PUFA productis separated from more polar components of the feed mixture.
 8. Theprocess according to claim 1, wherein components separated from the PUFAproduct in the second separation step comprise DHA or a DHA derivativeand/or other PUFAs or PUFA derivatives which are less polar than thePUFA product; and/or components separated from the PUFA product in thethird separation step comprise SDA or an SDA derivative and/or otherPUFAs which are more polar than the PUFA product.
 9. The processaccording to claim 1, wherein the eluent is a mixture of water and analcohol, an ether, an ester, a ketone or a nitrile.
 10. The processaccording to claim 9, wherein the eluent is a mixture of water andmethanol.
 11. The process according to claim 1, wherein the PUFA productcontains EPA or an EPA derivative in an amount greater than 95 wt %,preferably 97 wt %.
 12. The process according to claim 1, wherein theEPA derivative is EPA ethyl ester (EE).
 13. The process according toclaim 1, wherein a water:organic solvent ratio used in each separationstep is adjusted such that saturated and/or monounsaturated fatty acidspresent in the feed mixture are removed in the first separation step;and the PUFA product is separated from different components of the feedmixture in steps (ii) and (iii).
 14. The process according to claim 1,wherein the aqueous organic solvent eluent used in the second and thirdseparation steps has the same water:organic solvent ratio, and theaqueous organic solvent eluent used in the first separation step has adifferent water:organic solvent ratio from the organic solvent eluentused in the second and third separation steps, wherein the water:organic solvent ratio of the aqueous organic solvent eluent used in thefirst separation step is preferably lower than the water:organic solventratio of the aqueous organic solvent eluent used in the second and thirdseparation steps, and wherein the water:organic solvent ratio of theeluent used in the first separation step is more preferably from0.1:99.9 to 1:99 wt %, and the water:organic solvent ratio of the eluentused in the second and third separation steps is more preferably from7:93 to 9:91 wt %.
 15. The process according to claim 1, wherein part ofan extract or raffinate from the first, second or third step is recycledback into the apparatus.
 16. A computer program for controlling achromatography apparatus as defined in claim 1, which computer programcontains code means that, when executed, instructs the apparatus tocarry out a process according to claim 1.